Frozen tissue microarray technology for analysis of RNA, DNA, and proteins

ABSTRACT

The invention disclosed herein improves upon existing tissue microarray technology by using frozen tissues embedded in tissue embedding compound as donor samples and arraying the specimens into a recipient block comprising tissue embedding compound. Tissue is not fixed prior to embedding, and sections from the array are evaluated without fixation or post-fixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. Unlike paraffin tissue arrays which can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, the disclosed methods allow optimal evaluation by each technique and uniform fixation across the array panel. The disclosed arrays work well for DNA, RNA, and protein analyses, and have significant qualitative and quantitative advantages over existing methods.

FIELD OF THE INVENTION

This invention relates generally to tissue microarray technology.Microarray technology allows the rapid analysis of hundreds to thousandsof genes, mRNAs, proteins, and tissue samples in expedited experimentalapproaches and is used to identify and characterize genes and markersinvolved in a variety of human pathologies.

BACKGROUND OF THE INVENTION

Recently developed high density tissue microarray technology involvesarraying up to thousands of cylindrical tissue cores from individualtumors on a tissue microarray (see, e.g. Kononen et al. Nat Med. 1998July ;4(7):844-7). More than two hundred serial sections can then bemade from an individual microarray block and used for analysis of DNA,RNA, and/or proteins on a single glass slide. The technology is usefulin that it allows rapid analysis of a large number of samples so thatthe statistical relevance of new markers can be determined in a singleexperiment. In addition, altered expression levels can be correlated toamplification or deletion events in specific tumor samples using serialsections, allowing simultaneous determination of gene copy number andexpression analysis of candidate pathogenic genes and suppressor genes.Arrays have been made containing numerous tumor types (see, e.g. Schramlet al. Clin Cancer Res. 1999 August ;5(8):1966-75) as well as multiplestages and grades within individual tumor types (see, e.g. Moch et al.Am J Pathol. 1999 April ;154(4):981-6; Bubendorf et al. Cancer Res. Feb.15, 1999 (4):803-6 and Bubendorf et al. J Natl Cancer Inst. Oct. 20,1999 ;91(20):1758-64). This new technology has already proven useful forrapidly characterizing the prevalence and prognostic significance ofdifferentially expressed genes identified using cDNA array technology(see, e.g. Bubendorf et al. J Natl Cancer Inst. Oct. 20,1999;91(20):1758-64; Moch et al. Verh Dtsch Ges Pathol. 1999;83:225-32.German and Barlund et al. J Natl Cancer Inst. Aug. 20, 2000;92(15):1252-9) as well as genes involved in cancer development andprogression (see, e.g. Bubendorf et al. Cancer Res. Feb. 15,1999;59(4):803-6 and Bubendorf et al. J Natl Cancer Inst. Oct. 20,1999;91(20):1758-64). Tissue microarrays have also been useful inidentifying genes that are targets of chromosomal amplification (see,e.g. Barlund et al. Cancer Res. Oct. 1, 2000; 60(19):5340-4 and Richteret al. Am J Pathol. 2000 September ;157(3):787-94) as well as to studythe expression patterns of putative tumor suppressor genes (see, e.g.Bowen et al. Cancer Res. Nov. 1, 2000;60(21):6111-5).

A variety of technical problems exist with the current methodology,however, relating to the fact that the arrayed samples have to bepre-fixed and embedded in paraffin. The quality of the studies performedon sections from tissue array technology may be limited by the fixationmethods used on the original sample. Buffered formalin solutions (andrelated compounds) are among the most widely used tissue fixatives.These chemicals fix the tissue by acting as progressive cross linkersbetween proteins and nucleic acids, by introducing modifications in RNA(adding mono-methyl groups to its bases), and by producing coordinatebonds for calcium ions; these processes can damage RNA and alter targetantigenic structure by blocking or damaging antibody binding sites (see,e.g. Masuda et al. Nucleic Acids Res. Nov. 15, 1999;27(22):4436-43 andWerner et al. Am J Surg Pathol. 2000 July;24(7):1016-9. Review).Formalin fixation-induced alterations can make in-situ analysis of DNA,RNA, and proteins suboptimal and variations in the duration of fixationcan effect the quality and reproducibility of results (see, e.g. Kononenet al. Nat Med. 1998 July;4(7):844-7; Werner et al. Am J Surg Pathol.2000 July;24(7):1016-9, Review and Specht et al. Am J Pathol. 2001February;158(2):419-429). Artisans attempt to overcome fixation problemsfor FISH by uniformly pre-fixing tissues in cold ethanol and embeddingin paraffin (see, e.g. Kononen et al. Nat Med. 1998 July;4(7):844-7),but this approach is not optimal for array analysis of some proteins orfor RNA using in situ hybridization. Paraffin embedding of ethanol-fixedtissue does not prevent RNA degradation (see, e.g. Goldsworthy et al.Mol Carcinog. 1999 June;25(2):86-91). In addition, while ethanolfixation of tissue and subsequent paraffin embedding circumventsformalin fixation-related problems introduced by crosslinking, there arestill problems relating to the embedding, and/or deparaffinizationprocesses such as temperature-induced antigenic alterations introducedduring the embedding process (see, e.g. Werner et al. Am J Surg Pathol.2000 July;24(7):1016-9, Review; Battifora et al. J Histochem Cytochem.1986 August;34(8):1095-100 and Penault-Llorca et al. J Pathol. 1994May;173(1):65-75).

Consequently there is a need in the art to identify additional methodsthat allow for the optimal preservation of biological molecules such aspolypeptides and polynucleotides to be analyzed in such arrays. Thepresent invention meets this need in the art by providing methods thatcircumvent problems associated with traditional paraffin arrays.

SUMMARY OF THE PREFERRED EMBODIMENTS

The invention disclosed herein improves upon existing tissue microarraytechnology by arraying the specimens into a recipient block comprisingtissue embedding compound. Tissue is not fixed prior to embedding, andsections from the array are evaluated without fixation or post-fixedaccording to the appropriate methodology used to analyze a specific geneat the DNA, RNA, and/or protein levels.

The invention disclosed herein includes a number of embodiments. Atypical embodiment of the invention is method of preparing a tissuemicroarray by embedding a non-fixed biological sample in the tissuemicroarray block, wherein the tissue microarray block comprises frozentissue embedding compound. A related embodiment of the inventionincludes a method of preparing a tissue microarray comprising the stepsof: preparing a tissue microarray block for receipt of a biologicalsample, wherein the tissue microarray block comprises frozen tissueembedding compound; removing a core sample of a biological sample from afrozen donor block comprising tissue embedding compound; and thenplacing the core sample of the biological sample into an array withinthe tissue microarray block. Yet another embodiment of the invention isa method of preparing a biological sample for microarray analysiscomprising the steps of: preparing a tissue microarray block for receiptof a biological sample; freezing the biological sample in tissueembedding compound; removing a core sample of the biological sample fromthe frozen tissue embedding compound; and then placing the core sampleof the biological sample into an array within the tissue microarrayblock, wherein the tissue microarray block comprises frozen tissueembedding compound.

A number of variations on these methods are disclosed herein. Inpreferred embodiments for example, the biological sample is prepared forplacement into the tissue microarray block by removing a core sample offrozen biological material from a donor block comprising frozen tissueembedding compound. Preferably, the biological material is removed fromthe frozen tissue embedding compound with a coring means having atemperature of less than about 4 degrees centigrade. In yet anotherembodiment of the invention, a slice of an about 4 μm section of thefrozen tissue microarray block comprising a portion of the biologicalsample is removed for subsequent analysis. In such embodiments of theinvention, the tissue can be fixed after being embedded in the frozentissue embedding compound.

Another embodiment of the invention includes a process for preparing abiological sample for microarray analysis comprising embedding anon-fixed biological sample into an array within a block comprisingfrozen tissue embedding compound. A closely related embodiment includesa biological sample for microarray analysis prepared by this process.Embodiments of the invention also include compositions comprising anarray of biological samples having at least one non-fixed biologicalsample embedded in a tissue microarray block, wherein the tissuemicroarray block comprises frozen tissue embedding compound.

The invention also provides article of manufacture or kit comprising oneor more polypeptide and/or polynucleotide probes and a tissue embeddingmedium.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating preferredembodiments of the present invention, are given by way of illustrationand not limitation. Many changes and modifications within the scope ofthe present invention may be made without departing from the spiritthereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thepatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1: Frozen Microarray Method and HE staining. A) A total of 96 1.0mm samples from solid tumor mouse xenografts (derived from Calu-6, ahuman lung cancer cell line) spaced 1.0 mm apart are embedded in anO.C.T. block mounted on a plastic cassette as described in the Materialsand Methods. B) After the array is completed, a cylinder is mounted withO.C.T. to the back of the array which readily fits into the Hacker OTFcryostat for sectioning. C) A 4 micron section of the block shown in 1Ais HE-stained to show overall integrity and spacing, and D)4×magnification of the same section shows level of tissue and cellmorphology maintained in the OCT array.

FIG. 2: Non-radioactive RNA in situ hybridization. Non-radioactive RNAin situ hybridization with digoxigenin-labeled actin on frozen tissuemicroarray Calu-6 mouse xenograft sample at A) 20×magnification showsmRNA expression levels can be assessed using this technology. B)Negative control on a consecutive 4 micron section at 20×magnificationshows no signal.

FIG. 3: Fluorescent in Situ Hybridization (FISH). FISH on Calu-6 mousexenograft tissue microarray 4 micron section shown in FIG. 1 showsintense signals with a chromosome 8 centromere probe (Vysis). Signalsare easily detected on DAPI-counterstained nuclei at 100×magnificationwith a triple-pass filter on a fluorescent microscope.

FIG. 4: Immunohistochemistry. Antibody staining for the EGF receptor onfrozen array sample MDA-MB-231 (human breast cancer cell line known toexpress EGF receptor) shows at A) 4×magnification that staining isrelatively uniform and specific across the sample, and B) at 40Xmagnification shows expected membrane-specific staining when compared toC) no background staining on serial section with secondary antibodystaining only, and D) HE staining of the same sample from a serialsection of the array.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein ate intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The term “biological sample” is used herein according to its broadestmeaning and refers to the wide variety of biological materials that canbe analyzed in tissue microarrays. Biological materials typicallyanalyzed in tissue microarrays include tissues from specific organs suchas brain, kidney, liver heart, bone, prostate and other tissues, as wellas fluid materials such as serum, urine, semen etc. Such materials alsoinclude in vivo and in vitro cellular materials such as cancer cells andcell lines. The macromolecules analyzed in these materials typicallyinclude polypeptides such as proteins as well as polynucleotides such asRNA and DNA.

The term “tissue embedding compound” as used herein refers to artaccepted compounds artisans utilize to rapidly freeze biologicalmaterials for histopathological analysis, for example the medium sold byTissue Tek® under the name “O.C.T. (optimal cutting temperature)compound™” (product code 4583) and the medium sold by Instrumedics® Inc.under the name “Cryo-Gel™” (Cat#ICG-12). Tissue embedding compounds arecharacterized as being generally non-reactive with biological materialsand having a high degree of viscosity due to the presence of viscositygenerating substances such as polyvinyl alcohol and polyethylene glycol(see, e.g. O.C.T. compound which is composed of 10.24% polyvinylalcohol, 4.26% polyethylene glycol and 85.50% non-reactive ingredients).This definition excludes paraffin compounds typically used in tissuemicroarrays.

The terms “fixing” and “fixed” are used according to their art acceptedmeaning and refer to the chemical treatment (typically cross-linking) ofbiological materials such as proteins and nucleic acids that can beaccomplished by the wide variety of fixation protocols known in the art(see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 14,Frederick M. Ausubul et al. eds., 1995). The term “non-fixed” refers tobiological materials that have not been chemically modified or treated(e.g. with reagents such as formalin and ethanol) according to suchprotocols.

As noted above, microarray technology allows for the rapid analysis ofhundreds to thousands of genes, proteins, and other macromolecules inexpedited experimental approaches (see, e.g. Kononen et al. Nat Med.1998 July;4(7):844-7). This relatively new technology has already shownpotential in rapidly identifying and characterizing genes and markersinvolved in the pathogenesis of human cancers (see, e.g. Schraml et al.Clin Cancer Res. 1999 August;5(8):1966-75; Moch et al. Am J Pathol. 1999April;154(4):981-6; Bubendorf et al. Cancer Res. Feb. 15,1999;59(4):803-6; Bubendorf et al. J Natl Cancer Inst. Oct. 20,1999;91(20):1758-64; Moch et al. Verh Dtsch Ges Pathol. 1999;83:225-32.German and Barlund et al. J Natl Cancer Inst. Aug. 2,2000;92(15):1252-9; Barlund et al. Cancer Res. Oct. 1, 2000;60(19):5340-4; Richter et al. Am J Pathol. 2000 September;157(3):787-94and Bowen et al. Cancer Res. Nov. 1, 2000 ;60(21):6111-5). To date,human malignant tissue microarrays are most commonly constructed fromarchival paraffin tissue blocks. The paraffin-based technology may notbe optimal for studying RNA, DNA, and proteins simultaneously on asingle array because FISH, RNA in situ, and immunohistochemistry allhave different optimal fixation conditions.

The tissue microarrays described herein are made by modifying themethods used with traditional formalin-fixed paraffin-embeddedbiological samples. In such conventional methods, the sampling of theoriginal tissues for arraying is performed from morphologicallyrepresentative regions of regular formalin-fixed paraffin-embedded tumorblocks. Core tissue biopsies (diameter 0.6 mm, height 3-4 mm) are takenfrom individual “donor” blocks and arrayed into a new “recipient”paraffin block (45×20 mm) using a tissue microarraying instrument(Beecher Instruments Inc.). Typically artisans prefer cylinders rangingfrom about 0.6 to about 2.0 mm, which convey some histologicalinformation, yet allow up to one thousand specimens to be arrayed ineach block with little damage to the original blocks. The donor block ismanually positioned for sampling based on a visual alignment with thecorresponding HE-stained section on a slide. The region of interest forpunching in each tumor is carefully selected from the H&E stained slide.After the block construction is completed, about 4 to about 10 μmsections of the resulting tumor tissue microarray block are cut with amicrotome. An adhesive-coated tape sectioning system (Instrumedics Inc.)can be used for assisting sectioning of the tumor array blocks. Onaverage, two hundred sections can be cut from one tumor tissuemicroarray block. HE-staining for histology verification can beperformed periodically such as every 50th section cut from the block.Tissue microarray slides can be evaluated either manually or utilizing ahigh-throughput digital imaging system. For brightfield imageacquisition artisans typically utilize systems such as those produced byCarl Zeiss which ate based on high-resolution (4k×3k pixels) digitalcameras.

Technical problems with the existing methodology are related to the factthat the arrayed samples have been pre-fixed and embedded in paraffin.One way to avoid these problems and ensure optimal preservation ofantigens and nucleic acids is to use non-fixed (e.g. fresh frozen)tissue frozen at −70° C. (see, e.g. Slamon et al. Science. May 12,1989;244(4905):707-12 and Battifora et al. Am J Clin Pathol. 1991). Themethods disclosed herein overcome problems associated with paraffinarrays and demonstrate the feasibility of using frozen tissue forcreating tumor tissue microarrays.

The invention disclosed herein is represented by a number ofembodiments. A typical representative embodiment includes a method ofpreparing a tissue microarray comprising the steps of: preparing atissue microarray block for receipt of a biological sample; andembedding a non-fixed biological sample in the tissue microarray block,wherein the tissue microarray block comprises frozen tissue embeddingcompound. Typically the tissue microarray block is prepared for receiptof a biological sample by positioning it to receive this sample from asample dispensing means. In preferred embodiments of the invention, thebiological sample is prepared for placement into the tissue microarrayblock by removing a core sample of frozen biological material from adonor block that also comprises frozen tissue embedding compound. Whilethe donor and recipient blocks may comprise different compounds, inpreferred embodiments they are both formed from tissue embeddingcompound. In highly preferred embodiments of the invention, the coresample of the biological material is removed from the frozen tissueembedding compound with a coring means having a temperature of less thanabout 4 degrees centigrade (and preferably less than about −20°centigrade). Modifications to these methods include slicing an about 4μm section off of the frozen tissue microarray block; wherein thesection comprises a portion of the biological sample. In certainembodiments of the invention, the tissue is fixed after being embeddedin the frozen tissue embedding compound and sectioned from the block.

Preferred tissue embedding compounds include the medium sold by TissueTek® under the name “O.C.T. (optimal cutting temperature) compound™”(product code 4583) and the medium sold by Instrumedics® Inc. under thename “Cryo-Gel™” (Cat#ICG-12). These tissue embedding compounds functionto rapidly freeze biological samples and typically comprise viscosityagents such as about 5% to about 20% polyvinyl alcohol and/or about 1%to about 10% polyethylene glycol. In preferred embodiments of theinvention, the tissue embedding compound comprises about 10% polyvinylalcohol and about 4% polyethylene glycol.

Other embodiments of the invention include a method of preparing abiological sample for microarray analysis by freezing the biologicalsample in tissue embedding compound, removing a core sample of thebiological sample from the frozen tissue embedding compound, preparing atissue microarray block for receipt of a biological sample and thenplacing the core sample of the biological sample into an array withinthe tissue microarray block, wherein the tissue microarray blockcomprises frozen tissue embedding compound. In preferred aspects of theinvention, the core sample of the biological sample is removed from thefrozen tissue embedding compound with a coring means having atemperature of about −50° C. degrees centigrade. Modifications to thesemethods include slicing an about 4 μm section off of the frozen tissuemicroarray block; wherein the section comprises a portion of thebiological sample. In certain embodiments of the invention, the tissueis fixed after being embedded in the frozen tissue embedding compound.

Yet another embodiment of the invention includes a method of preparing atissue microarray comprising the steps of preparing a tissue microarrayblock for receipt of a biological sample, wherein the tissue microarrayblock comprises frozen tissue embedding compound, removing a core sampleof a biological sample from a frozen donor block comprising tissueembedding compound and then placing the core sample of the biologicalsample into an array within the tissue microarray block. In preferredaspects of the invention, the core sample of the biological sample isremoved from the frozen tissue embedding compound with a coring meanshaving a temperature of about −50° C. degrees centigrade. Modificationsto these methods include slicing an about 4 μm section off of the frozentissue microarray block; wherein the section comprises a portion of thebiological sample. In certain embodiments of the invention, the tissueis fixed after being embedded in the frozen tissue embedding compound.

Yet another embodiment of the invention is a process for preparing abiological sample for microarray analysis comprising embedding anon-fixed biological sample into an array within a block comprisingfrozen tissue embedding compound. A closely related aspect of thisembodiment is a biological sample for microarray analysis prepared bythis process. A highly preferred embodiment of the invention is acomposition comprising an array of biological samples comprising atleast one non-fixed biological sample embedded in a tissue microarrayblock, wherein the tissue microarray block comprises frozen tissueembedding compound. Specific illustrations of the various embodiments ofthe invention discussed in the preceding paragraphs ate provided below.

As disclosed in Example 1, test arrays (40×0.6 mm diameter samples and96×1.0 mm samples) using the methods disclosed herein were created. Thisexample demonstrates how samples can be cored from frozen tissue samples(and cell lines) embedded in O.C.T. compound (or directly thawed forcell lines) and placed into a frozen O.C.T. recipient array block forsectioning and subsequent storage. The standard 0.6 mm microarrayneedles (Beecher Instruments, Silver Spring, Mich.), which are used forthe paraffin-based microarrays, can core frozen tissue, but minimalpressure must be applied to prevent needle breakage. Typically, larger(1.0 mm) needles (Beecher Instruments, Silver Spring, Mich.) are farsturdier and preferred. Frozen tumor tissue and cell lines embedded inO.C.T. compound were successfully cored and placed into an O.C.T.compound recipient block. The array was constructed with≧1 mm spacebetween punches. A≧1 mm space between punches is preferred becausearraying samples more proximally may cause some cracking in therecipient array block. The frozen tissue array samples maintainedadequate morphology as seen when sections as thin as 4 microns were cutand HE stained (FIGS. 1C and 1D). A tape transfer system (InstrumedicsInc.) facilitated maintaining the integrity of the samples (comparearray in 1A to HE-stained slide made from section of 1A, shown in 1C).Similar results were obtained using a tape transfer system to section ahuman breast tumor array. The morphology and integrity of the humanbreast tumor array was comparable to the array shown in FIGS. 1C and 1Dproviding evidence that this technique will be successful when usingfatty tissues that are generally difficult to section using standardmethods (cryosectioning without the tape system).

In a typical test array, 96 samples (with 1 mm diameter) were easily fitin the array block with room to add more samples if needed (FIG. 1A).This sample size is equivalent to that seen in commercially availableparaffin arrays. For example, currently paraffin arrays containing up tosixty individual tissue samples (with 2 mm diameter) and up to twohundred individual tissue samples (with 0.6 mm diameter) can bepurchased from SuperBioChips Laboratories, Seoul, Korea, and InvitrogenCorp., San Diego, Calif., respectively. To fit more samples on thefrozen array, a larger mold (e.g. a plastic cryomold or a metal paraffinmold) can be used, or a smaller needle can be used to fit more samplesin the same space. The smaller needle biopsies have two considerations:(1) the smaller needles break more easily; and (2) there is lessrepresentation of the tumor the biopsy is derived from. One way toovercome problems with needle breakage problem is to let the frozentissue thaw a little before biopsying the sample. In the methodsdisclosed herein, briefly thawing frozen tissue did not effect test RNA(actin) quality.

To demonstrate how the tumor tissue microarray can be used for analysisof RNA, non-radioactive RNA in situ hybridization was performed on thetissue microarray slide using a digoxigenin labeled actin RNA probe. Asdisclosed in Example 2, array slides were fixed for 10 minutes, 2 hours,or overnight in 4% paraformaldehyde to test whether shorter fixationtimes could be used for non-radioactive RNA in situ hybridization. Usingactin as a probe, the studies demonstrated excellent preservation ofintact RNA when the array section was fixed overnight in 4%paraformaldehyde (FIG. 2). Slides fixed for 10 minutes and 2 hoursshowed no signal suggesting shorter fixation times may result inineffective fixation.

As disclosed in Example 3, methods employing a frozen tissue array arean excellent approach for FISH based experiments to analyze DNA (FIG.3). To demonstrate this, FISH of a chromosome 8 centromere probe to thefrozen tissue microarray was assayed to test whether the array could beused for in situ analysis of tumor DNAs. To determine which fixativeworks best for FISH to the frozen tissue microarray, array slides werefixed in either Carnoy's fixative or ethanol. A slightly stronger signalwas observed when slides for FISH were pre-fixed with Carnoy's fixativeas compared to ethanol fixation, but both worked well. Array slides werealso pretreated with and without proteinase K to determine whetherproteinase K treatment would have an effect on the quality ofhybridization and signal intensity. The proteinase K treatment did notimprove hybridization efficiency or signal intensity, as the probepenetrated the frozen tissue equally well under both conditions.

As disclosed in Example 4, immunohistochemical methods are easilyperformed with a frozen tissue array. To demonstrate this,immunohistochemistry on the tumor tissue microarray with antibodies forthe EGF receptor (HER-1) and heregulin was performed. The EGF receptorstaining is uniform across the sample (FIG. 4A) and gives the expectedmembrane-associated staining, as seen when comparing the EGFreceptor-stained sample with a serial HE-stained section (FIG. 4B,D).There is no background staining using the secondary antibody alone (FIG.4C). Similar results were obtained using the heregulin antibody exceptthe staining showed the expected diffuse cytoplasmic signal. Antibodiesto heregulin and the EGF receptor both result in cell-specific signalsshowing this methodology is useful for immunohistochemical based proteinanalyses as well.

The generation of a frozen tissue microarray comprising non-fixedtissues has a number of advantages over conventional practices. Forexample, such methods overcome problems associated with formalinfixation-induced alterations that effect the quality and reproducibilityof in-situ analysis of DNA, RNA, and proteins (see, e.g. Iononen et al.Nat Med. 1998 July;4(7):844-7; Werner et al. Am J Surg Pathol. 2000July;24(7):1016-9, Review and Specht et al. Am J Pathol. 2001February;158(2):419-429). In addition, by fixing a biological sampleafter the tissue microarray is constructed, one optimizes the study of aspecific macromolecule of interest by using the optimal fixingprotocol(s) that depend on the specific characteristics of that molecule(e.g. DNA, RNA, or proteins). In this way, different molecules ofinterest from a single tissue microarray can be evaluated in parallelsections under optimal conditions (e.g. a first section fixed for theevaluation of polynucleotides and a second section from the samemicroarray fixed for the evaluation of polypeptides). Other benefitsinclude the uniform fixation across the array panel that occurs when abiological sample is fixed after the tissue microarray is constructed(thereby decreasing signal variability that is associated withinconsistent fixing).

Frozen biological materials are typically difficult to manipulatewithout cracking or otherwise compromising the integrity of the frozenmatrix in which they are embedded. Consequently, a skilled artisan wouldexpect a frozen material to crack when exposed to the significantmechanical stresses associated with tissue microarray protocols. Suchmechanical stresses include those associated with the coring of themicroarray block (typically involving hundreds of punctures) as well asthe stresses associated with cutting about 4 to about 10 μm sections ofthe microarray block prior to their analysis. Consequently it issurprising that tissue microarrays can in fact be generated usingbiological materials placed within a frozen matrix such as tissueembedding medium. Moreover, because it is not possible to predict howthe freezing of a biological material in a specific medium and itssubsequent manipulation and analysis in that medium will effect thehistological data obtained in the context of a tissue microarray (e.g.the possibility that the frozen material will break apart whenmanipulated in this manner) the results described in Examples 2-4 areunexpected. In addition, the finding that tissue microarrays whichincorporate non-fixed biological materials produce results comparable towhich incorporate fixed biological materials is unexpected in view ofart which teaches the advantages of fixing biological materials inmicroarray protocols.

As demonstrated herein, frozen tissue microarrays provide excellenttarget material for the study of DNA, RNA, and proteins by fixing eacharray slide in a manner specific to the corresponding technique used.While there is some distortion of cell morphology and tissuearchitecture compared to formalin fixed paraffin embedded arrays, thisis commonly seen when comparing frozen sections to paraffin sections. Inaddition, skilled artisans will understand the need to modifytraditional methods when working with a frozen medium, for example thefact that the tissue embedding compound may bend and crack when samplesare placed at less than 1 mm apart.

A significant advantage of the frozen microarray approach stems from thefact that certain antibodies, DNA, and RNA probes do not performoptimally in pre-fixed paraffin embedded tissues. These reagents workvery well using the technology presented here. Another advantage of thefrozen tissue microarrays is that those procedures requiring fixationcan be conducted in samples fixed in an identical manner. Therefore, ahigher proportion of the arrayed samples may be included in the finalanalysis than with the paraffin embedded tumor microarrays. Asdemonstrated herein, frozen tumor tissue microarrays provide anexcellent way to store and analyze tumor samples and may prove usefulfor identifying novel molecular targets for diagnosis, prognosis, andtherapy of cancer, as well as for validation of cDNA microarray studies.By allowing simultaneous analysis of uniformly and optimally fixed DNA,RNA, and proteins from hundreds of tumor samples, this technologyfacilitates advances in the understanding of tumor pathobiology.

As is known in the art, one can utilize a variety of techniques known inthis art to enhance and/or improve upon microarray methods. Inparticular, one can employ and or modify methods from traditionalparaffin arrays to be used with the non-fixed frozen tissue arraysdisclosed herein (see, e.g. U.S. Pat. Nos. 6,103,518, 6,258,541 and6,251,601). In addition, variations on analyses include the variety ofmeans used in the art to optimize the interpretation of data such assuch as statistical sampling. For example skilled artisans can overcomechallenges associated with sampling error, by taking multiple biopsiesfrom each sample. A recent study of immunohistochemistry on paraffinarrays has shown that double sampling of 0.6 mm diameter punches oftumors leads to representation of the original tumor in at least 95% ofthe tumors on the array (see, e.g. Camp et al. Lab Invest 2000December;80(12):1943-9). Double punching may also improve representationusing the larger needles as well. Separate recipient arrays can be maderepresenting duplicate samples so that total sample number doesn't haveto be limited by double-punching.

For use in the methods described herein, kits are also within the scopeof the invention. Such kits can comprise a carrier, package or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method. For example, thecontainer(s) can comprise a probe that is or can be detectably labeled.Such probe can be an antibody or polynucleotide specific for a moleculeof interest. Where the method utilizes nucleic acid hybridization todetect the target nucleic acid, the kit can also have containerscontaining nucleotide(s) for identification of the target nucleic acidsequence and/or a container comprising a reporter-means, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, florescent, or radioisotopelabel.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including mediums and packageinserts with instructions for use. A label can be present on thecontainer to indicate that the composition is used for a specificapplication, and can also indicate directions for tissue use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit

A variety of modifications and improvements to the present inventionwill be apparent to those skilled in the art. While the descriptionabove refers to particular embodiments of the present invention, it willbe understood that many modifications may be made without departing fromthe spirit thereof. The accompanying claims are intended to cover suchmodifications as would fall within the true scope and spirit of thepresent invention. The presently disclosed embodiments are therefore tobe considered in all respects as illustrative and not restrictive, thescope of the invention being indicated by the appended claims, ratherthan the foregoing description, and all changes which come within themeaning and range of equivalency of the claims are therefore intended tobe embraced therein. All patent and patent application and literaturereferences cited in the present specification are hereby incorporated byreference in their entirety.

EXAMPLES Example 1 Frozen Array Construction

A human lung cancer cell line CALU-6 grown as a mouse xenograft andhuman breast cancer cells MDA-MB-231 grown in vitro and pelleted werefrozen and embedded in O.C.T. compound embedding medium (Miles Inc.Diagnostic Division, Elkhart, Ind.) to test whether arrays could becored and collected in this medium. Methodology was as publishedpreviously (1), except that tissue biopsies (diameter 0.6 and 1.0 mm;height 3-4 mm) were punched from tumors in O.C.T. and placed directlyinto an O.C.T. array block using a tissue microarrayer (BeecherInstruments, Silver Spring, Md., USA).

The recipient O.C.T. array block was made by filling a Tissue-Tekstandard cryomold (Miles Inc., Elkhart Ind.) with O.C.T. and mountingthe O.C.T. filled mold to the base of a plastic biopsy cassette (SimportHistosette II Biopsy Cassette from Fisher Scientific with lid removed),see FIG. 1A. The recipient O.C.T. block has the same size base as theparaffin recipient block that the tissue microarrayer was made toaccommodate, and therefore it was easily mounted in the tissuemicroarrayer (Beecher Instruments). The recipient block must besurrounded with dry ice to prevent melting. The same needle (0.6 or 1.0mm Beecher Instruments, Silver Spring, Mich., USA) was used for bothcoring the recipient array block and collecting the core biopsy ratherthan switching to a larger needle for the biopsies. The tissue in theneedle was kept frozen by holding the needle against a piece of dry icebefore and after punching the tissue and while dispensing the tissuecore into the recipient block. Punching and coring were done slowly withminimal pressure to prevent needle breakage. The recipient array waskept frozen by placing a piece of dry ice on its upper surface at alltimes except when punching and filling holes. A space of one millimeterwas left between each 0.6 or 1.0 mm punch.

Multiple arrays were created to demonstrate the feasibility of thismethod. One array contained 40 samples (0.6 mm in diameter). This arrayconsisted of 20 samples of a cell line (MDA-MB-231) frozen in O.C.T.(frozen cells quick-thawed and pipetted into a hole in the recipientO.C.T. block also work), and 20 biopsies of a solid tumor frozen inO.C.T. (Calu6 mouse xenografts) cored and placed in the recipient O.C.T.block. The second array contained 96 biopsies (1.0 mm in diameter) ofsolid tumors frozen in O.C.T. (Calu6 mouse xenografts), shown in FIG. 1.After the frozen tissue arrays were completed, a mounting cylinder(Hacker Instruments Inc., Fairfield, N.J., USA) was fixed with O.C.T.medium to the back of the array (FIGS. 1A and 1B). 4-10 micron sectionsof the whole block were cut from the array block using a cryostatmicrotome (Hacker Instruments Inc.) and the Basic Cryojane Tape TransferSystem and slides (Instrumedics Inc., Hackensack N.J.). The remainingtissue array was stored at −70° C. Slides were HE-stained to assess themorphological integrity of the tissue microarray (FIGS. 1C and 1D).

A tape transfer system (Instrumedics, Inc.) facilitates the preservationof the morphology and integrity of the array. As shown in FIGS. 1C and1D, HE staining was done on sections using the tape system and thefrozen tissue array samples maintains adequate morphology as seen whensections as thin as 4 micron were cut and HE stained (FIGS. 1C and 1D).The tape transfer system (Instrumedics Inc.) facilitates the maintenanceof the integrity of the samples (compare array in 1A to HE-stained slidemade from section of 1A, shown in 1C). Using the tape transfer systemwhen sectioning arrays, there was no problem sectioning a cell line(MDA-MB-231) or solid tumors (mouse xenografts derived from Calu6).Additionally, there was no problem sectioning frozen breast tissuearray. The morphology and integrity of the human breast tumor array wascomparable to the array shown in figure 1C and 1D providing evidencethat this technique is successful with tissues that are generallydifficult to section using standard methods.

Example 2 Non Radioactive RNA in situ Hybridization Methods EmployingFrozen Arrays

Nonradioactive RNA in situ hybridization was performed as publishedpreviously for frozen sections (see, e.g. Hogan et al. Manipulating theMouse Embryo: A Laboratory Manual. New York: Cold Spring HarborLaboratory Press (1994). Briefly, tissue array sections used for RNA insitu hybridization were fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS) for either 10 minutes, 2 hours, or overnight at 4C. Slides were rinsed 3 times in PBS for 5 minutes each and drained.Sections were covered with a prehybridization buffer (50% deionizedformamide, 5×SSC, 5×Denhardt's, 750 μg/ml torula RNA) and placed in ahumid chamber at room temperature for 2 hours. Hybridization wasperformed by adding 0.5 micrograms of digoxigenenin-labeled actin RNAprobe (Boehringer Mannheim, Germany) to 10 ml of prehybridizationsolution in a 5-slide mailer. Tissue nucleic acid was denatured at 85 Cfor 10 minutes and cooled on ice. The RNA probe was omitted as anegative control to determine background due to detection reagents.Slides were hybridized overnight at 70 C, then washed in 5×SSC at 70 Cfor 5 minutes and in 0.2×SSC at 70 C for 60 minutes. Sections were nextwashed for 5 minutes in buffer B1(0.1 M maleic acid, 0.15 M NaCl) andplaced in a humid chamber with blocking solution (1% Blocking agentBoehringer-Mannheim, Germany) for 1 hour at room temperature. Slideswere then drained and incubated at room temperature for 1 hour with a1:2000 dilution of AP conjugated alpha-digoxigenin antibody (RocheDiagnostics GmbH, Mannheim, Germany) in B2. The antibody was drained andslides were rinsed in B1 twice for 20 minutes each. Slides were nextwashed in B3 (100 mM Tris pH9.5, 100 mM NaCl, 5 mM MgCl2) for 5 minutes.Slides were then drained but not dried and covered with BM Purplesubstrate (Boehringer Mannheim GmbH, Germany) overnight. Signal waspost-fixed in 4% paraformaldehyde in PBS, and the signal visualizedusing standard light microscopy.

Typical results of such an assay are shown in FIG. 2.

Example 3 Fluorescent in situ Hybridization Methods Employing FrozenArrays

Frozen tissue microarray sections were fixed in Carnoy's fixative or 95%ethanol for 10 minutes. Slides were pretreated in 2×SSC at 37 C for 30minutes, dehydrated, denatured in 70% formamide/2×SSC for 5 minutes at72 C, and dehydrated again. Slides were then treated either with orwithout 0.4 ug/mil proteinase K (Sigma, St. Louis, Mo., USA) at 37 C for30 minutes. A spectrum orange chromosome 8 probe (Vysis Inc., Downer'sGrove, Ill.) was prepared according to the manufacturer's instructions,denatured for 7 minutes at 72 C, and hybridized to the array slidesovernight at 37 C in a humid chamber. Slides were washed (50% formanide/2×SSC 44 C, 15 minutes; 2×SSC, 8 minutes) and counterstained with DAPI(Vysis, Downersgrove, Ill., USA). Slides were visualized using standardfluorescent microscopy and photographed with Ektachrome 400 ASA slidefilm (Eastman Kodak, Rochester, N.Y., USA).

Typical results of such an assay are shown in FIG. 3.

Example 4 Immunohistochemical Methods Employing Frozen Arrays

Array slides for immunohistochemistry were prepared by sectioning of theblock as described above, then fixed in cold 100% methanol for 15minutes. Sections were rinsed in PBS, quenched in 0.45% hydrogenperoxide in PBS for 15 minutes and rinsed again. Immunohistochemistrywas performed using standard procedures (ABC-Elite, Vector Laboratories,Burlingame, Calif., USA). Briefly, slides were pre-incubated with normalgoat serum and blocking avidin for 20 minutes then rinsed in PBS.Monoclonal antibodies were used for detection of alpha-heregulin (SantaCruz Biotechnology, Santa Cruz, Calif., USA), and the EGF receptor (BDBiosciences, San Diego, USA) at a 1:100 dilution. Slides were incubatedwith the heregulin antibody and biotin in normal goat serum or with theEGF receptor antibody and biotin in normal horse serum for one hour andrinsed in PBS. The primary antibodies were not included in negativecontrol experiments. Slides were incubated with the secondary antibody(biotinylated anti rabbit IgG made in goat diluted 1:350 in normal goatserum or biotinylated anti mouse IgG made in horse diluted 1:50 innormal horse serum, Vector Laboratories, Burlingame, Calif.) for onehour. Solutions A and B (ABC-Elite, Vector Laboratories, Burlingame,Calif., USA) were added simultaneously for 30 minutes. Diaminobenzidinewas used as a chromogen and arrays were visualized and photographedusing standard light microscopy.

Typical results of such an assay are shown in FIG. 4.

What is claimed is:
 1. A method of preparing a tissue microarraycomprising die steps of: (a) preparing a frozen tissue embeddingcompound for receipt of one or more non-fixed biological samples bymaking an array of holes in the frozen tissue embedding compound; and(b) introducing the one or more non-fixed biological samples into thearray of holes in the frozen tissue embedding compound; so that thetissue microarray is prepared.
 2. The method of claim 1, wherein thearray of holes in the frozen tissue embedding compound are made with aneedle.
 3. The method of claim 1, wherein the method comprisesintroducing at least two or more non fixed biological samples into thearray of holes frozen tissue embedding compound.
 4. The method of claim1, wherein the tissue embedding compound comprises about 10% polyvinylalcohol and about 4% polyethylene glycol.
 5. The method of claim 1,wherein the one or more non-fixed biological samples is fixed afterbeing embedded in the frozen tissue embedding compound.
 6. The method ofclaim 1, further comprising the step of slicing an about 4 μm sectionoff of die frozen tissue embedding compound; wherein the sectioncomprises a portion of the embedded one or more non-fixed biologicalsample.